Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

Abstract

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.

Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription-Loop-Mediated Isothermal Amplification

“…Actually, RT-LAMP detected the rubella virus genome in all six samples positive for RT-PCR and also in one of three samples negative for RT-PCR. The detection limit of RT-LAMP for rubella (30 PFU/ml in culture fluids) showed the slightly lower sensitivity of this method compared with that for other viruses: 0.15 to 0.4 50% tissue culture infective doses (TCID 50 )/ml of measles virus (10), 3 PFU/ml of mumps virus (20), and 0.5 to 1.5 TCID 50 /ml of RSV (27). RT-LAMP for measles virus, mumps virus, and RSV has been used for routine clinical examinations in our laboratory.…”

“…The RT-PCR was performed with 1.25 U of Taq DNA polymerase (TaKaRa BioMedicals, Tokyo, Japan) in a thermal cycler (TaKaRa BioMedicals, Tokyo, Japan) with 35 rounds of the following thermal cycling conditions; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 min 40 seconds. The PCR products were confirmed by electrophoresis through a 1.5% agarose gel and stained with ethidium bromide (10,20,27).…”

Development of qRT-PCR Test for Quantification of Rubella Virus in Commercially Available Vaccines

“…The qRT-PCR is not only accurate but is also faster than the old clinical diagnostic methods (22). There is also no risk of contamination in this method and its sensitivity and specificity for the RNA determination in clinical specimens is high enough for accurate quantification (23,24). In this study, specific primers were designed, and PCR conditions were defined for efficient amplification and quantification of Rubella virus RNA.…”

Detection of Mumps Virus Vaccine Strain in Breast Milk After Postpartum Vaccination

“…Mumps viral LAMP primers were prepared based on the report of Okafuji et al 1) , and examined under the reaction conditions of 63℃ for 60 minutes. Nested RT-PCR was performed based on the report of Kidokoro et al…”